Summary of Work: Extensive genetic studies have established that the fidelities of replicative DNA polymerases and their associated proofreading 3-exonucleases are the primary determinants of mutation rates. Structural information is now available for many of these enzymes, prompting structure-driven mutational analyses of fidelity. We have developed a bacteriophage system that permits the rapid analysis of the fidelities of mutant polymerases in vivo without a requirement for enzyme over-expression, purification, and fidelity analysis in vivo. Bacteriophage RB69 DNA polymerase supplied from a plasmid can replace the normal T4 DNA polymerase when the latter is mutationally inactivated, retaining high fidelity during T4 DNA replication. Because the RB69 DNA polymerase can be crystallized whereas the T4 DNA polymerase cannot, only the structure of the RB69 enzyme is described. We are in the process of mutating critical polymerase and exonuclease amino acids and characterizing the resulting mutation rates using both reversion and forward-mutation tests. We have altered a key residue in the active site to produce a strong mutator polymerase that specifically produces transition mutations. We find that factors of increase caused by mutator mutations in vivo are much higher than those produced by the same mutations in vivo, and we are investigating a key difference in the way that a polymerase mutator and an exonuclease mutator interact in vitro versus in vivo. - Amino Acids, Bacteriophage, DNA, DNA Replication, Exonuclease, Genetic, Mutant, Polymerase